Preparation of templates for methylation analysis

ABSTRACT

The invention relates to a method of preparing and using a library of template polynucleotides suitable for use as templates in solid-phase nucleic acid amplification and sequencing reactions to determine the methylation status of the cytosine bases in the library. In particular, the invention relates to a method of preparing and analysing a library of template polynucleotides suitable for methylation analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication 60/900,313, filed Feb. 7, 2007. Applicants claim thebenefits of priority under 35 U.S.C. § 119 as to the ProvisionalApplication, the entire disclosure of which is incorporated herein byreference in its entirety.

This invention was made with government support under Grant No3-U54-HG003067-03S2, awarded by the National Institute of Health. TheUnited States government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to a method of preparing and using a library oftemplate polynucleotides suitable for use as templates in solid-phasenucleic acid amplification and sequencing reactions to determine themethylation status of the cytosine bases in the library. In particular,the invention relates to a method of preparing and analysing a libraryof template polynucleotides suitable for methylation analysis.

BACKGROUND TO THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Molecular biology and pharmaceutical drug development now make intensiveuse of nucleic acid analysis. The most challenging areas are wholegenome sequencing, single nucleotide polymorphism detection, screeningand gene expression monitoring.

In many eukaryotes, between 10% and 30% of cytosine bases are modifiedby the enzymatic addition of a methyl group to the 5 position of thebase. Although this modification does not interfere with the fidelity ofDNA replication processes, it enables modulation of diverse cellularprocesses through protein interactions with hypo- or hyper-methylatedsequences. These methylated sequences are not randomly dispersedthroughout a genome, but instead are almost exclusively found inrepetitive CpG sequences in regulatory regions upstream of many genes.Methylation of these sequences is associated with repression of geneactivity and can result in global changes to gene expression. Forexample, methylation plays a central role in the inactivation of one ofthe two X chromosomes in female cells, which is a prerequisite forensuring that females do not produce twice the level of X linked geneproducts as males. Methylation also underlies the selective repressionof either the maternally or paternally inherited copy of pairs ofalleles in a process known as genetic imprinting. It also silencestransposable elements whose expression would otherwise be deleterious toa genome.

Patterns of methylation in a genome are heritable because of thesemi-conservative nature of DNA replication. During this process, thedaughter strand, newly replicated on a methylated template strand is notinitially methylated, but the template strand directs methyltransferaseenzymes to fully methylate both strands. Thus methylation patterns carryan extra level of genetic information down through the generations inaddition to that information inherited in the primary sequence of thefour nucleotides.

Aberrant patterns of genomic methylation also correlate with diseasestates and are among the earliest and most common alteration found inhuman malignancies. Moreover, mistakes made during the establishment ofmethylation patterns during development underlie several specificinherited disorders. Consequently, there is a demand for high throughputapproaches for profiling the methylation status of many genes inparallel both for research purposes and for clinical applications.

Many methods already exist for detecting the methylation of DNA and theycan be broadly classified depending on the level of sequence-specificinformation they produce. On the simplest level, there are techniquesthat only yield information on overall levels of methylation within agenome. For example, methylated sequences can be separated fromunmethylated sequences on reverse-phase HPLC due to the difference inhydrophobicity of DNase I treated DNA. Such methods are simple but donot provide any information regarding the sequence context of themethylation sites. Alternatively, pairs of restriction endonucleasesthat recognize the same sequence but have different sensitivities tocytosine methylation at that sequence can be used. Methylation at thissequence will render it refractory to cleavage by one enzyme, butsensitive to the other. If no cytosine bases are methylated in asequence, both enzymes will produce identically sized restrictionfragments. In contrast, if methylation is present, the enzymes willproduce different sizes of fragments that can be distinguished bystandard analytical techniques such as electrophoresis through agarose.If Southern blot analysis is subsequently performed and the bands probedwith a labelled fragment from a gene of interest, then information onthe sequence context of the methylation site can be investigated. Thesemethods are limited because they are dependent on the availability ofuseful restriction enzymes and are confined to the study of methylationpatterns among sequences that contain those restriction sites.

5-Methylcytosine (5 mC) is a key epigenetic DNA modification inmammalian genomes. It occurs almost exclusively in the dinucleotidesequence mCpG and plays a central role in development and disease. Amongthe various methods for large-scale DNA-methylation analysis, onlybisulfite sequencing affords single CpG resolution. Bisulfite deaminatesunmethylated cytosine to uracil, while 5 mC is not affected. SequencingPCR-amplified bisulfite-converted DNA thus displays C and 5 mC as T andC, respectively.

Methods that do not rely on sequence context but which can detectmethylation at any chosen sequence are mainly based on the sodiumbisulfite reaction. Under controlled conditions, this reagent convertscytosine to uracil while methyl-cytosine remains unmodified. If thetreated DNA is then sequenced, the detection of a cytosine indicatesthat the cytosine is methylated because it would have been otherwiseconverted to a uracil.

Standard Sanger sequencing procedures have the disadvantage that only alimited number of sequencing reactions can be performed at the sametime. Moreover, PCR amplification and sub-cloning may be necessary toproduce sufficient quantities of DNA for sequencing, and both methodscan introduce artifacts into the sequence, including changes inmethylation.

Microarrays comprise molecular probes, such as nucleic acid molecules,arranged systematically onto a solid, generally flat surface or on acollection of beads or microspheres. Each probe site comprises a reagentsuch as a single stranded nucleic acid, whose molecular recognition of acomplementary nucleic acid molecule leads to a detectable signal, oftenbased on fluorescence. Microarrays comprising many thousands of probesites can be used to monitor gene expression profiles for a large numberof genes in a single experiment on a hybridisation based format.

Nucleic acid probes on microarrays are generally made in two ways. Acombination of photochemistry and DNA synthesis allows base-by-basesynthesis of the probes in situ. This is the approach pioneered byAffymetrix for growing short strands of around 25 bases. Their‘genechips’ are commercially available and widely used (e.g., Wodlickaet al., 1997, Nature Biotechnology 15:1359-1367), despite the expense ofmaking arrays designed for a particular experiment. Another method forpreparing microarrays is to use a robot to spot small (nL) volumes ofnucleic acid sequences onto discrete areas of the surface. Microarraysprepared in this manner have less dense features than Affymetrix arrays,but are more universal and cheaper to prepare (e.g., Schena et al.,1995, Science 270:467-470). The main drawback of all types of standardmicroarrays is the complex hardware required to achieve a spatialdistribution of multiple copies of the same DNA sequence.

WO 98/44151 and WO 00/18957 both describe methods of formingpolynucleotide arrays based on “solid-phase” nucleic acid amplification,which is analogous to a polymerase chain reaction wherein theamplification products are immobilised on a solid support in order toform arrays comprised of nucleic acid clusters or “colonies”. Eachcluster or colony on such an array is formed from a plurality ofidentical immobilised polynucleotide strands and a plurality ofidentical immobilised complementary polynucleotide strands. The arraysso-formed are generally referred to herein as “clustered arrays” andtheir general features will be further understood by reference to WO98/44151 or WO 00/18957, the contents of both documents beingincorporated herein in their entirety by reference.

As aforesaid, the solid-phase amplification methods of WO 98/44151 andWO 00/18957 are essentially a form of the polymerase chain (PCR)reaction carried out on a solid support. Like any PCR, these methodsrequire the use of forward and reverse amplification primers capable ofannealing to a template to be amplified. In the methods of WO 98/44151and WO 00/18957, both primers are immobilised on the solid support atthe 5′ end. Other forms of solid-phase amplification are known in whichonly one primer is immobilised and the other is present in free solution(Mitra, R. D and Church, G. M., Nucleic Acids Research, 1999, Vol. 27,No. 24).

In common with all PCR techniques, solid-phase PCR amplificationrequires the use of forward and reverse amplification primers whichinclude “template-specific” nucleotide sequences which are capable ofannealing to sequences in the template to be amplified, or thecomplement thereof, under the conditions of the annealing steps of thePCR reaction. The sequences in the template to which the primers annealunder conditions of the PCR reaction may be referred to herein as“primer-binding” sequences.

PCR amplification cannot occur in the absence of annealing of theforward and reverse primers to primer binding sequences in the templateto be amplified under the conditions of the annealing steps of the PCRreaction, i.e. if there is insufficient complementarity between primersand template. Some prior knowledge of the sequence of the template is,therefore, required before one can carry out a PCR reaction to amplify aspecific template. The user generally must know the sequence of at leastthe primer-binding sites in the template in advance so that appropriateprimers can be designed, although the remaining sequence of the templatemay be unknown. The need for prior knowledge of the sequence of thetemplate increases the complexity and cost of solid phase PCR of complexmixtures of templates, such as genomic DNA fragments.

Certain embodiments of the methods described in WO 98/44151 and WO00/18957 make use of “universal” primers to amplify templates comprisinga variable template portion that it is desired to amplify, flanked 5′and 3′ by common or “universal” primer binding sequences. The“universal” forward and reverse primers include sequences capable ofannealing to the “universal” primer binding sequences in the templateconstruct. The variable template portion may itself be of known, unknownor partially known sequence. This approach has the advantage that it isnot necessary to design a specific pair of primers for each template tobe amplified; the same primers can be used for amplification ofdifferent templates provided that each template is modified by additionof the same universal primer-binding sequences to its 5′ and 3′ ends.The variable template sequence can therefore be any DNA fragment ofinterest. An analogous approach can be used to amplify a mixture oftemplates, such as a plurality or library of template nucleic acidmolecules (e.g. genomic DNA fragments), using a single pair of universalforward and reverse primers, provided that each template molecule in themixture is modified by the addition of the same universal primer-bindingsequences.

Such “universal primer” approaches to solid-phase amplification areadvantageous since they enable multiple template molecules of the sameor different, known or unknown sequence to be amplified in a singleamplification reaction on a solid support bearing a single pair of“universal” primers. Simultaneous amplification of a mixture oftemplates of different sequences by PCR would otherwise require aplurality of primer pairs, each pair being complementary to each uniquetemplate in the mixture. The generation of a plurality of primer pairsfor each individual template is not a viable option for complex mixturesof templates.

Adaptors that contain universal priming sequences can be ligated ontothe ends of templates. The adaptors may be single-stranded ordouble-stranded. If double-stranded, they may have overhanging ends thatare complementary to overhanging ends on the template molecules thathave been generated with a restriction endonuclease. Alternatively, thedouble-stranded adaptors may be blunt ended, in which case the templatesare also blunt ended. The blunt ends of the templates may have beenformed during a process to shear the DNA into fragments, or they mayhave been formed by a “polishing” reaction, as would be well known tothose skilled in the art, or may have been treated to give a singlenucleotide overhang.

A single adaptor or two different adaptors may be used in a ligationreaction with templates. If a template has been manipulated such thatits ends are the same, i.e. both are blunt ended or both have the sameoverhang, then ligation of a single compatible adaptor will generate atemplate with that adaptor on both ends. However, if two compatibleadaptors, adaptor A and adaptor B, are used, then three permutations ofligated products are formed: template with adaptor A on both ends,template with adaptor B on both ends, and template with adaptor A on oneend and adaptor B on the other end. This last product is, under somecircumstances, the only desired product from the ligation reaction andconsequently additional purification steps are necessary following theligation reaction to purify it from the ligation products that have thesame adaptor at both ends.

The above-mentioned prior art methods have inherent shortcomings thatlimit their utility with respect to a variety of genome wide analyses.Accordingly, the method of the present invention expands the spectrum ofgenome wide analyses that can be performed.

SUMMARY OF THE INVENTION

The present invention is directed to a method that uses a singlemethylated adaptor in a ligation reaction to generate a library ofadaptor-target-adaptor polynucleotides for use in subsequent methylationanalyses. The presence of methylated adaptors in theseadaptor-target-adaptor polynucleotides facilitates treatment of suchpolynucleotides with bisulfite for the purposes of determiningmethylation status of cytosine bases in the target portion of theadaptor-target-adaptor polynucleotides. This directed investigation ofmethylation status of only the target portion of theadaptor-target-adaptor polynucleotides is made possible by the fact thatthe adaptors ligated onto the ends of the targets are fully methylatedand are, therefore, resistant to bisulfite induced alterations. Thus,any unmethylated cytosine bases present in the adaptor-target-adaptorpolynucleotides originate exclusively in the target nucleic acid portionof the polynucleotides and will be converted to uracils during bisulfitetreatment. This feature of the present invention, therefore, facilitatesdirected analysis of target sequence methylation status andidentification of the specific sequence context in which a methylatedcytosine is found in the target sequence. The method can, moreover, beapplied to preparing samples for amplification on a solid surface usingsurface-bound primer sequences, with no prior knowledge of the targetsequences. The invention is, therefore, applicable to analysis of themethylation status of all cytosine bases across a whole genome sample(genome-wide analysis), as well as to more specific applications onsmaller samples.

A first aspect of the invention relates to a method of analysingmethylation status of cytosine bases in a nucleic acid, comprising:

-   a. providing a sample of fragmented double stranded nucleic acid    target fragments derived from said nucleic acid;-   b. ligating universal adaptors to the fragmented double stranded    nucleic acid target fragments to produce adaptor-ligated double    stranded nucleic acid target fragments comprising identical nucleic    acid bases at each termini, wherein cytosine bases in said universal    adaptors are methylated and said universal adaptors comprise a    region of double stranded nucleic acids and at least one region of    single stranded nucleic acids;-   c. treating the adaptor-ligated double stranded nucleic acid target    fragments with a reagent that converts the non-methylated cytosine    bases to uracil to produce a treated sample of adaptor-ligated    double stranded nucleic acid target fragments;-   d. sequencing the treated adaptor-ligated double stranded nucleic    acid target fragments;-   e. analysing the sequences of the treated sample to determine which    cytosine bases were converted to uracil bases, thereby determining    the methylation status of the nucleic acid.

A second aspect of the invention relates to methods of amplifying thetreated samples. Thus, in a particular embodiment, the inventionprovides methods of amplifying the adaptor-ligated double strandednucleic acid target fragments comprising identical nucleic acid bases ateach termini.

A third aspect of the invention relates to methods for the solid-phasenucleic acid amplification of template polynucleotide molecules whichcomprises preparing a library of template polynucleotide molecules whichhave known sequences at their 5′ and 3′ ends using the method accordingto the first aspect of the invention and carrying out a solid-phasenucleic acid amplification reaction wherein said template polynucleotidemolecules are amplified.

In a fourth aspect the invention provides a kit for use in preparing a5′ and 3′ modified library of template polynucleotide moleculescomprising known sequences at their 5′ and 3′ ends, the kit comprisingmethylated adaptor polynucleotides and oligonucleotide primers capableof annealing to the methylated adaptor polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a)-(d) illustrate several examples of forked mismatch adaptorsfor use in the method of the invention, specifically depicting differentoverhanging or blunt end structures permissible at the “ligatable” endof the adaptor. FIG. 1( e) schematically illustrates the sequencecomponents of the two partially complementary strands (denoted oligo Aand oligo B) which form the universal forked adaptor when annealed.Oligo A and Oligo B are prepared with all the cytosine bases in themethylated form. The 5′ end of oligo B is complementary (COMP) to a partof the SEQ PRIMER sequence in oligo A. Oligo A includes a single “T”nucleotide overhang at the 3′ end. The 5′ end of oligo A isphosphorylated. P represents a phosphate group; X and Y representsurface capture functionalities.

FIG. 2 illustrates one embodiment of the method of the invention basedon use of the universal forked adaptors illustrated in FIG. 1. Thefragmented double stranded nucleic acid target fragments are ligated tothe forked adaptors and then split into two portions, one of which istreated with sodium bisulfite. Both portions are then amplified andsequenced to determine the differences between the treated and untreatedportions. FIG. 2( a) depicts the steps of fragmenting a complex samplesuch as genomic DNA to generate a plurality of target duplex fragments,ligation of the target duplex fragments to mismatch (forked) adaptors togenerate adaptor-template constructs and removal of unbound adaptors.The forked adaptor may include a biotin group at the 5′ end, which isnot ligated to the target fragment, to facilitate solid-phase capture ofthe adaptor-target constructs, e.g. onto streptavidin magnetic beads.FIG. 2( b) depicts an initial primer extension reaction in which primersare annealed to mismatch adaptor regions on each strand of anadaptor-target construct and extended to generate extension productscomplementary to each strand of the adaptor-target construct. Forsimplicity, the ligation and primer extension steps are illustrated fora single adaptor-target construct.

FIG. 3 illustrates an alternative embodiment of the invention in whichadaptor-target constructs are subjected to multiple rounds of primerannealing and extension to generate multiple single-stranded copies ofeach adaptor-target construct. For simplicity, the primer extensionsteps are illustrated for a single adaptor-target construct.

FIG. 4 illustrates a still further embodiment of the invention in whichadaptor-target constructs are subjected to PCR amplification to generatemultiple double-stranded copies of each adaptor-target construct. Forsimplicity, PCR amplification is illustrated for a single adaptor-targetconstruct.

FIG. 5 illustrates an embodiment of the invention, depicting steps offragmenting a complex sample such as genomic DNA to generate a pluralityof target fragments, ligation of the target fragments to mismatch(forked) adaptors to generate adaptor-template constructs and subsequentremoval of unbound adaptors, wherein the adaptors do not include abiotin group at the 5′ end. The resulting adaptor-target constructs maybe subjected to PCR amplification to generate multiple double-strandedcopies of each adaptor-target construct. For simplicity, the ligationsteps are illustrated for a single adaptor-target construct.

FIGS. 6( a)-(d) illustrate further examples of forked mismatch adaptorsfor use in the method of the invention, again depicting the permissibleblunt or overhang formats at the “ligatable” end of the adaptor. FIG. 6(e) schematically illustrates the component sequences present in the twostrands (denoted Oligo C and Oligo B) which form the adaptor whenannealed. Oligo B and Oligo C are prepared with all the cytosine basesin the methylated form. P represents a phosphate group; X and Yrepresent surface capture functionalities.

FIGS. 7( a)-(b) illustrate further embodiments of the invention based onuse of the forked adaptors illustrated in FIG. 6. FIG. 7( a) depictsfragmentation and ligation steps substantially similar to thoseillustrated in FIG. 5. FIG. 7( b) depicts subsequent PCR amplificationusing “tailed” PCR primers and schematically illustrates the sequencecomposition of the double-stranded amplification products formed in thePCR reaction. For simplicity, the ligation and PCR amplification stepsare illustrated for a single adaptor-target construct.

FIGS. 8( a)-(e) illustrate alternative embodiments of mismatch adaptorsfor use in the method of the invention wherein the single strandedregion takes the form of a ‘bubble’. The oligonucleotides D and E areprepared with the cytosine bases in the methylated form. P represents aphosphate group; X and Y represent surface capture functionalities; Wand Z represent modifications to prevent ligation.

FIGS. 9( a)-(b) illustrate further embodiments of the invention based onuse of the alternative adaptors illustrated in FIG. 8. FIG. 9( a)depicts fragmentation, ligation and subsequent removal of unboundadaptors. FIG. 9(b) depicts annealing of identical amplification primersto a duplex region of the adaptor on each strand of the adaptor-targetconstruct. The adaptor-target constructs can be amplified by PCR usingthis single primer species. For simplicity, the ligation steps andprimer annealing are illustrated for a single adaptor-target construct.

FIG. 10( a)-(c) illustrate an exemplary method of one embodiment of theinvention and the data generated thereby. The genomic DNA is fragmentedby limited digestion with a methylation-insensitive restriction enzymerather than by random hydrodynamic shearing. After size fractionation onan agarose gel, a narrow size window is isolated which constitutes onlya small portion of the genome. By careful size-selection, essentiallythe same genomic subfraction can be isolated from different inputsamples and compared by sequencing. In the exemplary embodimentillustrated in FIG. 10 a, genomic DNA from 4 different mouse cell typesis digested with the restriction enzyme MspI and size selected to 40-220bp resulting in a reduced representation of the mouse genome that isenriched for CpG dinucleotides and CpG islands. As shown in FIG. 10 b,the size selected fragments are equipped with the methylated forkedadapters, bisulfite converted and sequenced as described elsewhere inthis document. After mapping the bisulfite sequencing reads by aligningthem to the mouse reference genome, methylated cytosines are displayedas bisulfite-resistant cytosines (Cs). The MspI Reduced RepresentationBisulfite Sequencing approach has been used for comparative methylationprofiling of four different mouse cell types resulting in redundantcoverage of almost one million distinct CpG dinucleotides in each celltype with more than 800,000 CpGs covered in all four cell types (FIG. 10c).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention presents a method of analysing themethylation status of cytosine bases in a nucleic acid. The nucleic acidis fragmented, then ligated to universal adaptors wherein all thecytosine bases in the adaptors are methylated at the 5 position of thebase. The adaptors comprise two oligonucleotides which are partiallycomplementary such that they can hybridise to form a region of doublestranded sequence, but also retain a region of single stranded,non-hybridised sequence. A portion of the adaptor-target constructsample is treated to convert the unmethylated cytosine bases to uracil,and both the treated and untreated portions are sequenced to determinewhich cytosine bases in the target nucleic acid are methylated.

The ligation of universal adaptors to both ends of the target nucleicacid fragments gives rise to a pool of adaptor-ligated double strandednucleic acid target fragments with adaptors at both ends of the target.The treatment step to convert the non methylated cytosine bases touracil is usually performed with sodium bisulfite. After the treatment,the sample can be further amplified to produce a library of templatepolynucleotide molecules which have common sequences at their 5′ and 3′ends. In this context, the term “common” is interpreted as meaningcommon to all templates in the library, and is a known, artificiallyintroduced exogenous sequence that facilitates amplification of theentire library of template polynucleotide molecules. As explained infurther detail below, all templates within the library will containregions of known, common sequence at (or proximal to) their 5′ and 3′ends. The term library therefore refers to the collection of targetfragments containing known common sequences at their 3′ and 5′ ends, andmay also be referred to as a 3′ and 5′ modified library.

The library is formed by ligating identical adaptor polynucleotidemolecules (“universal adaptors”, the general features of which aredefined below) to the 5′ and 3′ ends of one or more fragmented doublestranded nucleic acid target fragments (which may be of known, partiallyknown or unknown sequence) to form adaptor-target constructs and thencarrying out an initial primer extension reaction in which extensionproducts complementary to both strands of each individual adaptor-targetconstruct are formed. The resulting primer extension products, andoptionally amplified copies thereof, collectively provide a library oftemplate polynucleotides.

The treatment with sodium bisulfite or similar reagent must be performedprior to any amplification steps in order to preserve the methylationstatus of the original sample. Once the bisulfite treatment has beenperformed, there is no need for subsequently utilised oligonucleotidesto be methylated. In other words, the common sequences in the amplifiedlibraries do not need to be derived from methylated amplificationprimers. The only sequences that need to be fully methylated are theadaptor sequences that are subjected to the bisulfite treatment.

Each strand of each template molecule in the library formed in theprimer extension reaction will therefore have the following structure,when viewed as a single strand:

5′-[common sequence I]-[target sequence]-[common sequence II]-3′wherein “common sequence I” represents a sequence derived from copying afirst strand of the universal adaptor and is common to all templatemolecules in the library generated in the initial primer extensionreaction; “target” represents a sequence derived from one strand of thefragmented double stranded nucleic acid target fragments, and may bedifferent in different individual template molecules within the library;and “common sequence II” represents a sequence derived from copying of asecond strand of the universal adaptor and is also common to alltemplate molecules in the library generated in the initial primerextension reaction.

Since “common sequence I” and “common sequence II” are common to alltemplate strands in the library they may include “universal”primer-binding sequences, enabling all templates in the library to beultimately amplified in a solid-phase amplification procedure usinguniversal primers.

It is a key feature of the invention, however, that the common 5′ and 3′end sequences denoted “common sequence I” and “common sequence II” arenot fully complementary to each other, meaning that each individualtemplate strand can contain different (and non-complementary) universalprimer sequences at its 5′ and 3′ ends.

To determine the methylation status of the nucleic acid, it is generallynecessary for libraries of templates to be amplified on a solid support,and ultimately sequenced. Amplified template molecules may thereforeinclude regions of “different” sequence at their 5′ and 3′ ends, whichare nevertheless common to all template molecules in the library. Forexample, the presence of a common unique sequence at one end only ofeach template in the library can provide a binding site for a sequencingprimer, enabling one strand of each template in the amplified form ofthe library to be sequenced in a single sequencing reaction using asingle type of sequencing primer.

Typically “common sequence I” and “common sequence II” will consist ofno more than 100, or no more than 50, or no more than 40 consecutivenucleotides at the 5′ and 3′ ends, respectively, of each strand of eachtemplate polynucleotide. The precise length of the two sequences may ormay not be identical. The nucleotide sequences of “common sequence I”and “common sequence II” in the template polynucleotides will bedetermined in part by the sequences of the adaptor strands ligated tothe target polynucleotides and in part by the sequence of the primerused in the initial primer extension reaction, and any subsequent roundsof nucleic acid amplification.

Additional sequences may be included at the 5′ end of “common sequenceII” in the amplified products, for example, by the use of “tailed” PCRprimers. In embodiments where the amplification is performed using a“tailed” amplification primer that extends beyond the 5′ end of theadaptor sequence, then the products of the amplification reaction willbe double-stranded polynucleotides, one strand of which has thestructure:

5′-[common sequence I]-[target sequence]-[common sequence II]-3′

It will be appreciated that “common sequence II” in the amplificationproducts may differ somewhat to the “common sequence II” present in theproducts of the primer extension using the shorter primers, since theformer will be determined solely by the sequence of the ligated adaptor,whereas the latter will be determined by both the adaptor sequence plusthe overhanging sequence of the amplification primers that can be copiedduring the amplification cycles. Nevertheless, since the PCR primer isdesigned to anneal to a sequence in the initial extension products whichis complementary to the 3′ adaptor, the two forms of “common sequenceII” will contain identical sequences at the 3′ end.

The precise nucleotide sequences of the common regions of the templatemolecules in the library are generally not material to the invention andmay be selected by the user. The common sequences must at least comprise“primer-binding” sequences which enable specific annealing ofamplification primers when the templates are in use in a solid-phaseamplification reaction. The primer-binding sequences are thus determinedby the sequence of the primers ultimately used for solid-phaseamplification. The sequence of these primers, in turn, is advantageouslyselected to avoid or minimise binding of the primers to the targetportions of the templates within the library under the conditions of theamplification reaction, but is otherwise not particularly limited. Byway of example, if the target portions of the templates are derived fromhuman genomic DNA, then the sequences of the primers used in solid phaseamplification should ideally be selected to minimise non-specificbinding to any human genomic sequence.

The universal adaptor polynucleotides used in the method of theinvention must contain a region of both double and single strandedsequence, i.e. they must not be formed by annealing of fullycomplementary polynucleotide strands. Such adaptors are defined as‘mismatched’ or ‘mismatch’ adaptors as long as they contain at least onestrand that is single stranded.

Mismatch adaptors for use in the invention can be formed by annealingtwo partially complementary polynucleotide strands so as to provide,when the two strands are annealed, at least one duplex region and atleast one single stranded region. The single stranded region in saidadaptors is defined as the “mismatch” region.

The “duplex region” of the adaptor is a short double-stranded region,typically comprising 5 or more consecutive base pairs, formed byannealing of the two partially complementary polynucleotide strands.

Generally it is advantageous for the duplex region to be as short aspossible without loss of function. By “function” in this context ismeant that the duplex region forms a stable duplex under standardreaction conditions for an enzyme-catalysed nucleic acid ligationreaction, which are known to the skilled reader (e.g. incubation at atemperature in the range of from 4° C. to 25° C. in a ligation bufferappropriate for the enzyme), such that the two strands forming theadaptor remain partially annealed during ligation of the adaptor to atarget molecule. It is not absolutely necessary for the duplex region tobe stable under the conditions typically used in the annealing steps ofprimer extension or PCR reactions.

Since identical adaptors are ligated to both ends of each fragmenteddouble stranded nucleic acid target fragment, the target sequence ineach adaptor-target construct will be flanked by complementary sequencesderived from the duplex region of the adaptors. The longer the duplexregion, and hence the complementary sequences derived therefrom in theadaptor-target constructs, the greater the possibility that theadaptor-target construct is able to fold back and base-pair to itself inthese regions of internal self-complementarity under the annealingconditions used in primer extension and/or PCR. Generally it ispreferred for the duplex region to be 20 or fewer, 15 or fewer, or 10 orfewer base pairs in length in order to reduce this effect. The stabilityof the duplex region may be increased, and its length potentiallyreduced, by the inclusion of non-natural nucleotides which exhibitstronger base-pairing than standard Watson-Crick base pairs.

In a particular embodiment, the two strands of the adaptor are 100%complementary in the duplex region. It will be appreciated, however,that one or more mismatched nucleotides may be tolerated within theduplex region, provided that the two strands are capable of forming astable duplex under standard ligation conditions.

Adaptors for use in the invention will generally include a duplex regionadjacent to the “ligatable” end of the adaptor, i.e. the end that isjoined to a target polynucleotide in the ligation reaction. Theligatable end of the adaptor may be blunt or, in other embodiments,short 5′ or 3′ overhangs of one or more nucleotides may be present tofacilitate/promote ligation. The 5′ terminal nucleotide at the ligatableend of the adaptor should be phosphorylated to enable phosphodiesterlinkage to a 3′ hydroxyl group on the target polynucleotide.

The term “mismatch region” refers to a region of the adaptor wherein thesequences of the two polynucleotide strands forming the adaptor exhibita degree of non-complementarity such that the two strands are notcapable of annealing to each other under standard annealing conditionsfor a primer extension or PCR reaction. The two strands in the mismatchregion may exhibit some degree of annealing under standard reactionconditions for an enzyme-catalysed ligation reaction, provided that thetwo strands revert to single stranded form under annealing conditions.

The conditions encountered during the annealing steps of a PCR reactionare generally known to one skilled in the art, although the preciseannealing conditions will vary from reaction to reaction (see Sambrooket al., 2001, Molecular Cloning, A Laboratory Manual, 3rd Ed, ColdSpring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY;Current Protocols, eds Ausubel et al.). Typically, such conditions maycomprise, but are not limited to, (following a denaturing step at atemperature of about 94° C. for about one minute) exposure to atemperature in the range of 50° C. to 65° C. (preferably 55-58° C.) fora period of about 1 minute in standard PCR reaction buffer, (optionallysupplemented with 1M betaine and 1.3% DMSO). Different annealingconditions may be used for a single primer extension reaction notforming part of a PCR reaction (again see Sambrook et al., 2001,Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor Laboratory Press, NY; CurrentProtocols, eds Ausubel et al.).

It is to be understood that the ‘mismatch region’ is provided bydifferent portions of the same two polynucleotide strands which form thedouble-stranded region(s). Mismatches in the adaptor construct can takethe form of one strand being longer than the other, such that there is asingle stranded region on one of the strands, or a sequence selectedsuch that the two strands do not hybridise, and thus form a singlestranded region on both strands. Adaptors used in this particularexample are termed ‘forked adaptors’. The mismatches may also take theform of ‘bubbles’, wherein both ends of the adaptor construct(s) arecapable of hybridising to each other and forming a duplex, but thecentral region is not. The portion of the strand(s) forming the mismatchregion does not anneal under conditions in which other portions of thesame two strands are annealed to form one or more double-strandedregions. For avoidance of doubt, it is to be understood that asingle-stranded or single base overhang at the 3′ end of apolynucleotide duplex that subsequently undergoes ligation to the targetsequences does not constitute an ‘mismatch region’ in the context ofthis invention.

The portions of the two strands forming the mismatch region typicallycomprise at least 10, or at least 15, or at least 20 consecutivenucleotides on each strand. The lower limit on the length of themismatch region will typically be determined by function, for example,the need to provide a suitable sequence for binding of a primer forprimer extension, PCR and/or sequencing. Theoretically there is no upperlimit on the length of the mismatch region, except that in general it isadvantageous to minimise the overall length of the adaptor, for example,in order to facilitate separation of unbound adaptors fromadaptor-target constructs following the ligation step. Therefore, it ispreferred that the mismatch region should be fewer than 50, or fewerthan 40, or fewer than 30, or fewer than 25 consecutive nucleotides inlength on each strand.

The portions of the two forked adaptor strands forming the mismatchregion should preferably be of similar length, although this is notabsolutely essential, provided that the length of each portion issufficient to fulfil its desired function (e.g. primer binding).

In a particular embodiment, the portions of the two forked adaptorstrands forming the mismatch region will be completely mismatched, or100% non-complementary. However, skilled readers will be appreciate thatsome sequence “matches”, i.e. a lesser degree of non-complementarity maybe tolerated in this region without affecting function to a materialextent. As aforesaid, the extent of sequence mismatching ornon-complementarity must be such that the two strands in the mismatchregion remain in single-stranded form under annealing conditions asdefined above.

The precise nucleotide sequence of the adaptors is generally notmaterial to the invention and may be selected by the user such that thedesired sequence elements are ultimately included in the commonsequences of the library of templates derived from the adaptors, forexample, to provide binding sites for particular sets of universalamplification primers and/or sequencing primers. Additional sequenceelements may be included, for example, to provide binding sites forsequencing primers which will ultimately be used in sequencing oftemplate molecules in the library, or products derived from solid-phaseamplification of the template library. The adaptors, or amplificationprimers may further include “tag” sequences, which can be used to tag ormark template molecules derived from a particular source. The generalfeatures and use of such tag sequences is described in applicant'spending application published as WO 05/068656, the contents of which areincorporated herein by reference in its entirety.

Although the precise nucleotide sequence of the methylated adaptor isgenerally non-limiting to the invention, the sequences of the individualstrands in the mismatch region of the forked adaptors should be suchthat neither individual strand exhibits any internalself-complementarity which could lead to self-annealing, formation ofhairpin structures, etc. under standard annealing conditions.Self-annealing of a strand in the mismatch region is to be avoided as itmay prevent or reduce specific binding of an amplification primer tothis strand.

The universal adaptors are preferably formed from two strands of DNA,but may include mixtures of natural and non-natural nucleotides (e.g.one or more ribonucleotides) linked by a mixture of phosphodiester andnon-phosphodiester backbone linkages. Other non-nucleotide modificationsmay be included such as, for example, biotin moieties, blocking groupsand capture moieties for attachment to a solid surface, as discussed infurther detail below. The biotin moieties may be used to effectisolation and removal of any unligated target fragments from theligation reaction.

The one or more “target polynucleotide duplexes” to which the adaptorsare ligated may be any polynucleotide molecules that it is desired toamplify by solid-phase PCR, generally with a view to sequencing. Thetarget polynucleotide duplexes may originate in double-stranded DNA form(e.g. genomic DNA fragments) or may have originated in single-strandedform, as DNA or RNA. The sample can not have been copied by a polymeraseprior to analysis, otherwise the methylation status of the sample willnot be maintained. Any bisulfite treatment must be carried out on theoriginal sample prior to any copying or amplification steps such asreverse transcription or PCR amplification. The precise sequence orsource of the target molecules is generally not material to theinvention, and may be known or unknown, and the methodology describedherein is applicable to the methylation analysis of the genome of anybiological organism.

The method of the invention may be applied to multiple copies of thesame target molecule (so-called monotemplate applications) or to amixture of different target molecules which differ from each other withrespect to nucleotide sequence over all or a part of their length. Themethod may be applied to a plurality of target molecules derived from acommon source, for example, a library of genomic DNA fragments derivedfrom a particular individual or organism. In one embodiment, the targetpolynucleotides comprise fragments of genomic DNA, which may be human.The fragments may be derived from a whole genome or from part of agenome (e.g. a single chromosome or sub-fraction thereof), and from oneindividual or several individuals. Techniques for fragmentation ofgenomic DNA, for example, by chemical or enzymatic digestion ormechanical shearing, sonication or nebulisation are encompassed by thepresent invention. The fragmentation may be random, for example usinghydro-dynamic shearing or nebulisation such that the ends of thefragments have random sequences. Alternatively the nucleic acid samplemay be treated with an enzyme such as a restriction endonuclease suchthat the ends of the fragments all comprise the same sequence. Theenzyme may recognise certain sequences for example those containing highlevels of C and G bases. The enzyme may select for CpG dinucleotides orCpG islands, for example MspI, whose recognition site is 5′-CCGG-3′ andcuts to give a 3′GC overhang on each fragment.

“Ligation” of adaptors to the 5′ and 3′ ends of each fragmented doublestranded nucleic acid target fragment involves joining of the twopolynucleotide strands of the adaptor to the double-stranded targetpolynucleotide such that covalent linkages are formed between bothstrands of the two double-stranded molecules. Preferably such covalentlinking takes place by formation of a phosphodiester linkage between thetwo polynucleotide strands but other means of covalent linkage (e.g.non-phosphodiester backbone linkages) may be used. However, it is anessential requirement that the covalent linkages formed in the ligationreactions allow for read-through of a polymerase, such that theresultant construct can be copied in a primer extension reaction usingprimers which bind to sequences in the regions of the adaptor-targetconstruct that are derived from the adaptor molecules.

The ligation reactions will preferably be enzyme-catalysed. The natureof the ligase enzyme used for enzymatic ligation is not particularlylimited. Non-enzymatic ligation techniques (e.g. chemical ligation) mayalso be used, provided that the non-enzymatic ligation leads to theformation of a covalent linkage which allows read-through of apolymerase, such that the resultant construct can be copied in a primerextension reaction.

The desired products of the ligation reaction are adaptor-targetconstructs in which universal, methylated adaptors are ligated at bothends of each target polynucleotide, given the structureadaptor-target-adaptor. Conditions of the ligation reaction shouldtherefore be optimised to maximise the formation of this product, inpreference to targets having an adaptor at one end only.

The products of the ligation reaction may be subjected to purificationsteps in order to remove unbound adaptor molecules before theadaptor-target constructs are processed further. Any suitable techniquemay be used to remove excess unbound adaptors, particular examples ofwhich will be described in further detail below.

Following bisulfite treatment, adaptor-target constructs formed in theligation reaction may be subjected to an amplification reaction in whicha primer oligonucleotide is annealed to an adaptor portion of each ofthe adaptor-target constructs and extended by sequential addition ofnucleotides to the free 3′ hydroxyl end of the primer to form extensionproducts complementary to at least one strand of each of theadaptor-target constructs.

The primers used for the amplification reaction will be capable ofannealing to each individual strand of adaptor-target constructs havingadaptors ligated at both ends, and can be extended so as to obtain twoseparate primer extension products, one complementary to each strand ofthe construct. Thus, in a particular embodiment, the initial primerextension reaction results in formation of primer extension productscomplementary to each strand of each adaptor-target

In a particular embodiment, the primer used in the initial primerextension reaction anneals to a primer-binding sequence (in one strand)in the mismatch region of the adaptor. The primer may also hybridise tothe double stranded region of the adaptor. If the adaptor contains a3′-overhanging base complementary to an overhanging base in the targetsequence, then the amplification primers may also hybridise to theligated target region of the fragmented double stranded nucleic acidtarget fragments. Such amplification primers may be beneficial inhelping to reduce the amplification of any adaptor dimers which maycontaminate the sample preparation.

The term “annealing” as used in this context refers to sequence-specificbinding/hybridisation of the primer to a primer-binding sequence in anadaptor region of the adaptor-target construct under the conditions usedfor the primer annealing step of the initial primer extension reaction.

The products of the primer extension reaction may be subjected tostandard denaturing conditions in order to separate the extensionproducts from strands of the adaptor-target constructs. Optionally thestrands of the adaptor-target constructs may be removed at this stageif, for example, the adaptors contain a biotin sequence that can beselectively bound using an avidin or streptavidin bead. The extensionproducts (with or without the original strands of the adaptor-targetconstructs) collectively form a library of template polynucleotideswhich can be used as templates for solid-phase PCR.

If desired, only a single amplification primer can be added to theamplification mixture, and the initial primer extension reaction may berepeated one or more times, through rounds of primer annealing,extension and denaturation, in order to form multiple copies of the sameextension products complementary to the adaptor-target constructs.

In other embodiments the initial extension products may be amplified byconventional solution-phase PCR, as described in further detail below.In a particular embodiment, both primers used for PCR amplificationanneal to different primer-binding sequences on opposite strands in themismatch region of the forked adaptor. Other embodiments may, however,be based on the use of a single type of amplification primer whichanneals to a primer-binding sequence in the duplex region of theadaptor. The amplification conditions also allow for amplification withmore than two primers if desired to carry out nested PCR and control thelength of the sequences added to the target fragments.

Inclusion of the initial primer extension step, and optionally furtherrounds of PCR amplification, to form complementary copies of theadaptor-target constructs prior to solid-phase PCR is advantageous, forseveral reasons. Firstly, inclusion of the primer extension step, andsubsequent PCR amplification, acts as an enrichment step to select foradaptor-target constructs with adaptors ligated at both ends. Onlytarget constructs with adaptors ligated at both ends provide effectivetemplates for solid-phase PCR using common or universal primers specificfor primer-binding sequences in the adaptors, hence it is advantageousto produce a template library comprising only double-ligated targetsprior to solid-phase ligation.

Secondly, inclusion of the initial primer extension step, and subsequentPCR amplification, permits the length of the common sequences at the 5′and 3′ ends of the target to be increased prior to solid-phase PCR. Asoutlined above, it is generally advantageous for the length of theadaptor molecules to be kept as short as possible, to maximise theefficiency of ligation and subsequent removal of unbound adaptors.However, for the purposes of solid-phase PCR it may be an advantage tohave longer sequences of common or “universal” sequences at the 5′ and3′ ends of the templates to be amplified. Inclusion of the primerextension (and subsequent amplification) steps means that the length ofthe common, known sequences at one (or both) ends of the polynucleotidesin the template library can be increased after ligation by inclusion ofadditional sequences at the 5′ ends of the primers used for primerextension (and subsequent amplification). The use of such “tailed”primers is described in further detail below.

Various non-limiting specific embodiments of the method of the inventionare described in further detail with reference to the accompanyingdrawings. Features described as being preferred in relation to onespecific embodiment of the invention apply mutatis mutandis to otherspecific embodiments of the invention unless stated otherwise.

FIG. 1 illustrates several embodiments of a particular type of mismatchadaptor for use in the method of the invention. The adaptor is formed byannealing two single-stranded oligonucleotides, herein referred to as“oligo A” and “oligo B”. Oligo A and oligo B may be prepared byconventional automated oligonucleotide synthesis techniques in routineuse in the art. The cytosine bases in oligonucleotides A and B must bemethylated at the 5 position of the base. Such oligonucleotides can beprepared according to standard procedures, from phosphoramidites inwhich the cytosine base is methylated. The oligonucleotides arepartially complementary such that the 3′ end of oligo A is complementaryto the 5′ end of oligo B. The 51 end of oligo A and the 3′ end of oligoB are not complementary to each other. When the two strands areannealed, the resulting structure is double stranded at one end (theduplex region) and single stranded at the other end (the mismatchregion) and is referred to herein as a “forked adaptor” (FIG. 1 a). Theduplex region of the forked adaptor may be blunt-ended (FIG. 1 b) or itmay have an overhang. In the latter case, the overhang may be a 3′overhang (FIG. 1 c) or a 5′ overhang (FIG. 1 d), and may comprise asingle nucleotide or more than one nucleotide.

The 5′ end of the double-stranded part of the forked adaptor isphosphorylated, i.e. the 5′ end of oligo B (FIG. 1 a-d). The presence ofthe 5′ phosphate group identifies this as the “ligatable” end of theadaptor. The 5′ end of oligo A may be biotinylated or bear anotherfunctionality (represented by X) that enables it to be captured on asurface, such as a bead. Alternative functionalities other than biotinare known to those skilled in the art. The 3′ end of oligo B may also bebiotinylated or bear another functionality (represented by Y) thatenables it to be captured on a surface (FIG. 1 d).

The phosphodiester bonds that comprise the back-bone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. Preferably only the last, or last andpenultimate, phosphodiester bonds at both the 3′ and 5′ ends of theoligonucleotides will be substituted with phosphorothioate bonds. In aparticular embodiment of the invention, oligo A contains a biotin groupon its 5′ end, oligo B is phosphorylated at its 5′ end and thedouble-stranded portion of the duplex contains a single base 3′ overhangcomprising a ‘T’ nucleotide. Oligo A consists of two regions: a regionat the 5′ end which is identical to a region of an amplification primerto be used for PCR amplification, referred to herein as “PRIMER 1”sequence, and at its 3′ end a region identical to that of a universalsequencing primer, referred to herein as “SEQ PRIMER” sequence, plus anadditional ‘T’ nucleotide on the 3′ end. Oligo B also consists of tworegions: a region at its 5′ end that is complementary to only part ofthe 3′ end of the SEQ PRIMER sequence in Oligo A, excluding the ‘T’overhang of Oligo A, and a region complementary to that of a universalPCR amplification primer, herein referred to as “comp-PRIMER 2” at its3′ end (FIG. 1 e).

FIG. 2 illustrates one embodiment of the method of the invention basedon use of the forked adaptors illustrated in FIG. 1. A mixture of targetDNA molecules of different sequence may be prepared by mixing a number,greater than one, of individual DNA molecules. In an aspect of theinvention, genomic DNA is fragmented into small molecules, less than1000 base pairs, more particularly less than 500 base pairs, and mostparticularly between 100-200 base pairs. Fragmentation of DNA may beachieved by a number of methods including: enzymatic digestion, chemicalcleavage, sonication, nebulisation, or hydroshearing, preferablynebulisation.

Fragmented DNA may be rendered blunt-ended by a number of methods knownto those skilled in the art. In a particular method, the ends of thefragmented DNA are “polished” with T4 DNA polymerase and Klenowpolymerase, a procedure well known to skilled practitioners, and thenphosphorylated with a polynucleotide kinase enzyme. A single ‘A’deoxynucleotide is then added to both 3′ ends of the DNA molecules usingTaq polymerase or Klenow exo minus polymerase enzyme, producing aone-base 3′ overhang that is complementary to the one-base 3′ ‘T’overhang on the double-stranded end of the forked adaptor.

A ligation reaction between the forked adaptor and the DNA fragments isthen performed using a suitable ligase enzyme (e.g. T4 DNA ligase) whichjoins two copies of the adaptor to each DNA fragment, one at either end,to form adaptor-target constructs. The products of this reaction can betreated immediately with sodium bisulfite before any furtherpurification or amplification steps. The ligated sample may be splitsuch that the treated and untreated portions can be compared, althoughif a reference sequence is known, then the whole sample or a portionthereof can be treated, sequenced and compared against the referencewithout the need to sequence an untreated version of the sample. Theanalysis of the nucleic acid sequences can be performed either bycomparing against the known reference or against a treated sample forthe purpose of determining which cytosine bases have been converted touracil bases due to the treatment step. The bisulfite treated sample isfurther treated to remove the sodium bisulfite to prevent contaminationof the untreated or untreated amplified sample. Both portions may becombined after bisulfite treatment and work-up, or separateamplification steps may be performed.

An oligonucleotide, herein referred to as PRIMER 2, which hybridises tothe “comp-PRIMER 2” sequence on the oligo B strand of the adaptor-targetconstructs can be used in an initial primer extension reaction togenerate a complementary copy of the adaptor-target strand. Anoligonucleotide, herein referred to as PRIMER 1, which hybridises to thesequence produced by extension of primer 2, can be used to enable astandard two primer amplification reaction. The library produced by theamplification reaction can be used directly, or further purified, foruse in sequencing to determine the differences between the treated anduntreated samples.

FIG. 2 a shows a ligation reaction between a biotinylated forked adaptorand the DNA fragments performed using a suitable ligase enzyme (e.g. T4DNA ligase) which joins two copies of the adaptor to each DNA fragment,one at either end, to form adaptor-target constructs. The products ofthis reaction can be purified from unligated adaptor by a number ofmeans, including size-inclusion chromatography, preferably byelectrophoresis through an agarose gel slab followed by excision of aportion of the agarose that contains the DNA greater in size than thesize of the adaptor.

After the excess adaptor has been removed, unligated target DNA remainsin addition to ligated adaptor-target constructs and this can be removedby selectively capturing only those target DNA molecules that haveadaptor attached. The presence of a biotin group on the 5′ end of OligoA of the adaptor enables any target DNA ligated to the adaptor to becaptured on a surface coated with streptavidin, a protein thatselectively and tightly binds biotin. Streptavidin can be coated onto asurface by means known to those skilled in the art. In a particularmethod, commercially available magnetic beads that are coated instreptavidin can be used to capture ligated adaptor-target constructs.The application of a magnet to the side of a tube containing these beadsimmobilises them such that they can be washed free of the unligatedtarget DNA molecules (FIG. 2 a). If desired, the bisulfite treatment canbe performed on the immobilised sample, to allow the bisulfite to beeasily removed from the treated adaptor-target constructs.

An oligonucleotide, herein referred to as PRIMER 2, which hybridises tothe “comp-PRIMER 2” sequence on the oligo B strand of the adaptor-targetconstructs can be used in an initial primer extension reaction togenerate a complementary copy of the adaptor-target strand attached tothe bead. The resulting primer extension product forms a double-strandedduplex with its complementary adaptor-target strand attached to the beadand it can then be isolated and purified from its complementaryadaptor-target strand on the bead by denaturation (FIG. 2 b).

There are several standard methods for separating the strand of a DNAduplex by denaturation, including thermal denaturation, or preferablychemical denaturation in either 100 mM sodium hydroxide solution orformamide solution. The pH of a solution of single-stranded DNA in asodium hydroxide solution collected from the supernatant of a suspensionof magnetic beads can be neutralised by adjusting with an appropriatesolution of acid, or preferably by buffer-exchange through asize-exclusion chromatography column pre-equilibrated in a bufferedsolution. The resulting solution contains a library of single-strandedDNA template molecules all of which comprise in order: 5′ PRIMER 2sequence, target DNA fragment, the complement of SEQ PRIMER sequence,then the complement of PRIMER 1 sequence. This template library can thenbe used on a solid-phase PCR platform that contains immobilised PRIMER 1and PRIMER 2 oligonucleotides, or can be further amplified in solutionusing primer 1 and primer 2.

FIG. 3 illustrates an alternative embodiment of the invention in whichadaptor-target constructs prepared as described above with reference toFIG. 2 b are subjected to multiple rounds of primer annealing andextension to generate multiple single-stranded copies of eachadaptor-target construct. In this embodiment of the invention, theinitial primer extension reaction on the bead immobilisedadaptor-template molecules with PRIMER 2 is in effect replaced with anasymmetric PCR amplification with the PRIMER 2 oligonucleotide (FIG. 3),this being equivalent to multiple rounds of the same primer extensionreaction. In this embodiment, multiple single-stranded copies of thebead-immobilised strands are generated in the supernatant of the beadsuspension due to PCR thermocycling, hence a separate denaturation stepis not necessary to recover the newly synthesised complementary copiesof the bead-immobilised adaptor-target strands; the copies can bepurified from the supernatant by standard methods, known to thoseskilled in the art.

In another embodiment of the invention, illustrated in FIG. 4, theinitial primer extension reaction on the bead-immobilised adaptor-targetconstructs with PRIMER 2 forms part of a standard (symmetric) PCRamplification with the PRIMER 2 and PRIMER 1 oligonucleotides. In thisembodiment, multiple double-stranded copies of the bead-immobilisedstrands are generated in the supernatant of the bead suspension due toPCR thermocycling, hence a separate denaturation step is not necessaryto recover the newly synthesised complementary copies of thebead-immobilised adaptor-target strands; the copies can be purified fromthe supernatant by standard methods, known to those skilled in the art.

In another embodiment of the invention, the adaptors are removed priorto amplification, as illustrated in FIG. 5. The forked adaptor does notcontain a biotin group at the 5′ end of the Oligo A strand. In thisembodiment, fragmented DNA may be made blunt-ended by a number ofmethods known to those skilled in the art. In a particular method, theends of the fragmented are polished with T4 DNA polymerase and Klenowpolymerase, and then phosphorylated with polynucleotide kinase enzyme. Asingle ‘A’ deoxynucleotide is then added to both 3′ ends of the DNAmolecules with Taq polymerase or Klenow exo minus polymerase enzyme,producing a one-base 3′ overhang that is complementary to the one-base3′ ‘T’ overhang on the double-stranded “ligatable” end of the forkedadaptor. A ligation reaction between the forked adaptor and the DNAfragments is then performed, e.g. using T4 DNA ligase enzyme, whichjoins two copies of the adaptor to each DNA template molecule, one ateither end.

The products of the ligation reaction can be purified from unligatedadaptor by a number of means, including size-inclusion chromatography,preferably by electrophoresis through an agarose gel slab followed byexcision of a portion of the agarose that contains DNA greater in sizethan the size of the adaptor. An aliquot of the purified template DNA isthen bisulfite treated as in FIG. 2, and used in a PCR amplificationwith the PRIMER 2 and PRIMER 1 oligonucleotides as described in FIG. 2.The first PCR cycle will involve an initial primer extension reactionwith primer 2 (not illustrated). The primers selectively amplify thosetemplate DNA molecules that have adaptors ligated on both ends. Theproduct of the reaction is a library of double-stranded templatemolecules, each of which comprise in order on one of the duplex strands:5′ PRIMER 2 sequence, target DNA (template fragment), the complement ofSEQ PRIMER sequence, then the complement of PRIMER 1 sequence. Thislibrary can then be amplified on a solid-phase PCR platform thatcontains immobilised PRIMER 1 and PRIMER 2 oligonucleotides, andcompared with sequences derived from the portion of the sample that hasnot been bisulfite treated.

FIG. 6 illustrates further examples of forked mismatch adaptors for usein the method of the invention. In this embodiment the forked adaptor isformed by annealing two single-stranded oligonucleotides, hereinreferred to as “oligo C” and “oligo B”. Both oligo B and oligo C need tohave the cytosine bases methylated at the 5 position of the base. Theoligonucleotides are partially complementary such that the 3′ end ofoligo C is complementary to the 5′ end of oligo B. The 5′ end of oligo Cand the 3′ end of oligo B are not complementary to each other. When thetwo oligos are annealed the resulting structure is double-stranded atone end (duplex region) and single-stranded at the other end (mismatchregion) (FIG. 6 a). The duplex region of the forked adaptor may beblunt-ended (FIG. 6 d) or it may have an overhang. In the latter case,the overhang may be a 3′ overhang (FIG. 6 c) or a 5′ overhang (FIG. 6b), and may comprise a single base or more than one base.

The 5′ end of the duplex region of the forked adaptor is phosphorylatedi.e. the 5′ end of ‘oligo B’ (FIGS. 6 a-d) to provide a “ligatable” end.The 5′ end of oligo C may be biotinylated or bear another functionality(X) that enables it to be captured on a surface, such as a bead. The 3′end of oligo B may also be biotinylated or bear another functionality(Y) that enables it to be captured on a surface (FIG. 6 d).

The phosphodiester bonds that comprise the back-bone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. Preferably only the last, or last andpenultimate, phosphodiester bonds at both the 3′ and 5′ ends of theoligonucleotides will be substituted with phosphorothioate bonds. OligoC comprises the following: a sequence identical to that of a universalsequencing primer denoted “SEQ PRIMER” (or identical to part of the 3′end of the “SEQ PRIMER” sequence), plus an additional ‘T’ nucleotide onthe 3′ end. Oligo B comprises the following regions: a region at its 5′end that is complementary to a part of the 3′ end of the SEQ PRIMERsequence in Oligo C, excluding the ‘T’ overhang of ‘Oligo C’, and aregion at its 3′ end which is complementary to that of a PCRamplification primer, herein referred to as the “comp-PRIMER 2”sequence, (FIG. 6 e).

FIG. 7 illustrates a further embodiment of the invention based on use ofthe forked adaptors illustrated in FIG. 6. In this embodiment,adaptor-target constructs are prepared substantially as described abovewith reference to FIG. 2 (FIG. 5 without the adaptor removal), exceptthat the adaptors illustrated in FIG. 6 are used (FIG. 7 a). Again, aportion of the sample (i.e., a subportion or part of the sample) istreated with bisulfite, and the resultant bisulfite treated andbisulfite untreated (control) portions are processed for sequencing.

As used herein, the term “untreated” or “control” portion of a samplerefers to a portion of the sample not exposed to the indicatedtreatment. The term may, therefore, be used to refer to a portion of asample that has not been treated with, for example, bisulfite, butrather has been exposed to or incubated in an appropriate control bufferthat is essentially inert with respect to bisulfite induced activity.

Each portion of the sample is used in a standard solution-phase PCRamplification with “tailed” primer oligonucleotides. Tailed primers areprimers that only hybridize via their 3′ end to a target sequence,leaving a 5′ non-hybridised tail. When used in amplifications by PCR,the initial round of PCR amplification (i.e. the first and second primerextension reactions) rely on binding of the 3′ ends of the tailedprimers to cognate primer-binding sequences in the adaptor regions ofthe adaptor-target constructs. The 5′ non-hybridising tails derived fromthe tailed primers act as templates in subsequent PCR cycles and aretherefore copied into the resultant double-stranded PCR products.

In the present embodiment, either one or both of the primers used in theamplification reaction can be “tailed” primers. In one embodiment, theprimers used are denoted PRIMER 3 and PRIMER 4, where PRIMER 3 consistsof a 5′ tail sequence, and a 3′ sequence that is complementary to the“comp PRIMER 2” sequence in the forked adaptor; and PRIMER 4 consists ofa 5′ tail sequence, and a 3′ sequence that is identical to the 5′ end ofthe SEQ PRIMER sequence present in the mismatch region of the forkedadaptor. Following amplification by PCR, the tail sequences areincorporated into the copies of the adaptor-target DNA construct.

In one embodiment of the invention, the tail sequences on PRIMER 3 andPRIMER 4 are non-identical sequences. The sequence ofsurface-immobilised primers to be used on a subsequent solid-phase DNAamplification platform can then be designed based on the tail sequenceof PRIMER 3 and the tail sequence of PRIMER 4 (FIG. 7 b).

In another embodiment of the invention, the tail sequences on PRIMER 3and PRIMER 4 are identical sequences. The products of the solution-phasePCR will thus have the same sequence at their ends, namely the commontail sequence of PRIMER 3 and PRIMER 4. This common tail sequence canthen be used as the basis on which to design the sequence of a singlesurface-immobilised primer on a solid-phase DNA amplification platform.Subsequent surface amplification of the library of templates may thus beperformed using a single PCR primer immobilised on the surface.

FIG. 8 illustrates alternative embodiments of mismatch adaptors for usein the method of the invention, wherein the mismatch region takes theform of a bubble. These “modified” forked adaptors may be designed toenable solid-phase amplification of templates using a single surfacebound primer. The adaptor is formed by annealing two single-strandedoligonucleotides, herein referred to as “oligo D” and “oligo E”. Botholigonucleotides D and E are modified such that all the cytosine basesare methylated at the 5 position. The oligonucleotides are partiallycomplementary such that the 3′ end of oligo D is complementary to the 5′end of oligo E, and the 5′ end of oligo D is complementary to the 3′ endof oligo E, however, the central portions of oligo D and oligo E arenon-complementary. When oligo D and oligo E are annealed, the resultingstructure is double stranded at both ends (duplex regions) and singlestranded in the middle (mismatch bubble region) and is referred toherein as the “modified Forked adaptor” (FIG. 8 a).

One end of the modified forked adaptor is modified to prevent ligationof a DNA molecule to this end. Such modifications are known to thoseskilled in the art. The other “ligatable” end may be blunt-ended (FIG. 8d) or may have an overhang. In the latter case, the overhang may be a 3′overhang (FIG. 8 c) or a 5′ overhang (FIG. 8 b), and may comprise asingle base or more than one base. The 5′ strand of the ligatable end isphosphorylated i.e. the 5′ end of oligo E (FIGS. 8 a-d). The 5′ end ofoligo D may be biotinylated or bear another functionality that enablesit to be captured on a surface, such as a bead. The 3′ end of oligo Emay be biotinylated or bear another functionality that enables it to becaptured on a surface (FIG. 8 d). The modifications to prevent ligation(Z,W) may be the same as or different to the surface capturefunctionalities (X,Y).

The phosphodiester bonds that comprise the backbone of theoligonucleotides may be replaced with non-enzymatically cleavable bondssuch as phosphorothioate bonds. In a particular embodiment, only thelast, or last and penultimate, phosphodiester bonds at both the 3′ and5′ ends of the oligonucleotides are substituted with phosphorothioatebonds.

In a particular embodiment of the invention, oligo E is phosphorylatedat its 5′ end and the 3′ end of oligo D contains a single base 3′overhang comprising a “T” nucleotide. Oligo D comprises two sequences: asequence at its 5′ end which is identical to that of a universalamplification primer, referred to herein as “PRIMER 5” sequence, next toa sequence identical to that of a universal sequencing primer denoted“SEQ PRIMER” sequence plus the additional “T” nucleotide on the 3′ end.Oligo E comprises three sequences: a sequence at its 5′ end that iscomplementary to only part of the 3′ end of the SEQ PRIMER sequence inOligo D, excluding the ‘T’ overhang of Oligo D, a central sequencenon-complementary to any part of Oligo D, and a 3′ end that iscomplementary to the “PRIMER 5” sequence of Oligo D (FIG. 8 e).

FIG. 9 illustrates a still further embodiment of the invention based onuse of the alternative adaptors illustrated in FIG. 8. In thisembodiment adaptor-target constructs may be prepared substantially asdescribed above in relation to FIG. 5, except that the modified forkedadaptors illustrated in FIG. 8 are used. The adapters can be removed anda portion of the sample treated with bisulfite. Both the bisulfitetreated and untreated aliquots of the adaptor-target constructs are usedin a solution-phase PCR amplification using PRIMER 5 oligonucleotide toselectively amplify those ligation products that have the modifiedadaptor on both ends (FIG. 9 b). The product of the solution-phase PCRcan then be purified and amplified on a solid-phase platform with asingle immobilised primer, e.g. PRIMER 5. Inclusion of the mismatchbubble sequence in oligo E ensures that all products of this solid-phaseamplification will contain common sequencing primer binding sequences onone strand only, enabling sequencing using a universal sequencing primerwhich anneals to this common sequence.

Solid-Phase Amplification

Once formed, the library of templates prepared according to the methodsdescribed above can be used for solid-phase nucleic acid amplification.

Thus, in further aspects the invention provides a method of solid-phasenucleic acid amplification of template polynucleotide molecules whichcomprises preparing a library of template polynucleotide molecules whichhave known sequences at their 5′ and 3′ ends using a method according tothe first aspect of the invention described herein and carrying out asolid-phase nucleic acid amplification reaction wherein said templatepolynucleotide molecules are amplified.

The term ‘solid-phase amplification’ as used herein refers to anynucleic acid amplification reaction carried out on or in associationwith a solid support such that all or a portion of the amplifiedproducts are immobilised on the solid support as they are formed. Inparticular, the term encompasses solid-phase polymerase chain reaction(solid-phase PCR) and solid phase isothermal amplification which arereactions analogous to standard solution phase amplification, exceptthat one or both of the forward and reverse amplification primers is/areimmobilised on the solid support. Solid phase PCR covers systems such asemulsions, wherein one primer is anchored to a bead and the other is infree solution, and colony formation in solid phase gel matrices whereinone primer is anchored to the surface, and one is in free solution.

The invention encompasses “solid-phase” amplification methods in whichonly one amplification primer is immobilised (the other primer usuallybeing present in free solution), as well as the solid support to beprovided with both the forward and the reverse primers immobilised. Inpractice, there will be a “plurality” of identical forward primersand/or a “plurality” of identical reverse primers immobilised on thesolid support, since the amplification process requires an excess ofprimers to sustain amplification. References herein to forward andreverse primers are to be interpreted accordingly as encompassing a“plurality” of such primers unless the context indicates otherwise.

As will be appreciated by the skilled reader, any given amplificationreaction requires at least one type of forward primer and at least onetype of reverse primer specific for the template to be amplified. Incertain embodiments, however, the forward and reverse primers maycomprise template-specific portions of identical sequence, and may haveentirely identical nucleotide sequence and structure (including anynon-nucleotide modifications). In other words, it is possible to carryout solid-phase amplification using only one type of primer, and suchsingle-primer methods are encompassed within the scope of the invention.Other embodiments may use forward and reverse primers which containidentical template-specific sequences but which differ in some otherstructural features. For example one type of primer may contain anon-nucleotide modification which is not present in the other.

In other embodiments of the invention the forward and reverse primersmay contain template-specific portions of different sequence.

In all embodiments of the invention, amplification primers forsolid-phase amplification are preferably immobilised by covalent singlepoint attachment to the solid support at or near the 5′ end of theprimer, leaving the template-specific portion of the primer free forannealing to its cognate template and the 3′ hydroxyl group free forprimer extension. Any suitable covalent attachment means known in theart may be used for this purpose. The chosen attachment chemistry willdepend on the nature of the solid support, and any derivatisation orfunctionalisation applied thereto. The primer itself may include amoiety, which may be a non-nucleotide chemical modification, tofacilitate attachment. In a particular embodiment, the primer mayinclude a sulphur-containing nucleophile, such as phosphorothioate orthiophosphate, at the 5′ end. In the case of solid-supportedpolyacrylamide hydrogels (as described below), this nucleophile willbind to a bromoacetamide group present in the hydrogel. A particularmeans of attaching primers and templates to a solid support is via 5′phosphorothioate attachment to a hydrogel comprised of polymerisedacrylamide and N-(5-bromoacetamidylpentyl) acrylamide (BRAPA), asdescribed in WO05065814, the contents of which are included herein byreference in their entirety.

Certain embodiments of the invention may make use of solid supportscomprised of an inert substrate or matrix (e.g. glass slides, polymerbeads, etc) which has been “functionalised”, for example by applicationof a layer or coating of an intermediate material comprising reactivegroups which permit covalent attachment to biomolecules, such aspolynucleotides. Examples of such supports include, but are not limitedto, polyacrylamide hydrogels supported on an inert substrate such asglass. In such embodiments, the biomolecules (e.g. polynucleotides) maybe directly covalently attached to the intermediate material (e.g. thehydrogel), but the intermediate material may itself be non-covalentlyattached to the substrate or matrix (e.g. the glass substrate). The term“covalent attachment to a solid support” is to be interpretedaccordingly as encompassing this type of arrangement.

The library of templates prepared according to the first aspect of theinvention can be used to prepare clustered arrays of nucleic acidcolonies, analogous to those described in WO 00/18957 and WO 98/44151,by solid-phase amplification. The terms “cluster” and “colony” are usedinterchangeably herein to refer to a discrete site on a solid supportcomprised of a plurality of identical immobilised nucleic acid strandsand a plurality of identical immobilised complementary nucleic acidstrands. The term “clustered array” refers to an array formed from suchclusters or colonies. In this context the term “array” is not to beunderstood as requiring an ordered arrangement of clusters.

The term solid phase, or surface, is used to mean either a planar arraywherein primers are attached to a flat surface, for example, glass,silica or plastic microscope slides or similar flow cell devices; beads,wherein either one or two primers are attached to the beads and thebeads are amplified; or an array of beads on a surface after the beadshave been amplified.

Clustered arrays can be prepared using either a process ofthermocycling, as described in patent WO9844151, or a process wherebythe temperature is maintained as a constant, and the cycles of extensionand denaturing are performed using changes of reagents. Such isothermalamplification methods are described in patent application numbersWO0246456 and US20080009420 (WO07107710) (Isothermal methods forcreating clonal single molecule arrays) and the contents of thesedocuments is included herein by reference. The lower temperaturesrequired in the isothermal process render this approach particularlyadvantageous.

Use in Sequencing/Methods of Sequencing

The invention also encompasses methods of sequencing amplified nucleicacids generated by solid-phase amplification. Thus, the inventionprovides a method of nucleic acid sequencing comprising amplifying alibrary of nucleic acid templates using solid-phase amplification asdescribed above and carrying out a nucleic acid sequencing reaction todetermine the sequence of the whole or a part of at least one amplifiednucleic acid strand produced in the solid-phase amplification reaction.As will be apparent to the skilled reader, references herein to aparticular nucleic acid sequence may, depending on the context, alsorefer to nucleic acid molecules which comprise the nucleic acidsequence. Sequencing of a target fragment means that a read of thechronological order of bases is established. The bases do not need to becontiguous, nor does every base on the entire fragment have to besequenced.

Sequencing can be carried out using any suitable sequencing technique,wherein nucleotides are added successively to a free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the nucleotide added is preferably determinedafter each nucleotide addition. Sequencing techniques using sequencingby ligation, wherein not every contiguous base is sequenced, andtechniques such as massively parallel signature sequencing (MPSS) wherebases are removed from, rather than added to the strands on the surfaceare also within the scope of the invention, as are techniques usingdetection of pyrophosphate release (pyrosequencing). Such pyrosequencingbased techniques are particularly applicable to sequencing arrays ofbeads wherein the beads have been amplified in an emulsion such that asingle template from the library molecule is amplified on each bead.

The initiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of the solid-phaseamplification reaction. In this connection, one or both of the adaptorsadded during formation of the template library may include a nucleotidesequence which permits annealing of a sequencing primer to amplifiedproducts derived by whole genome or solid-phase amplification of thetemplate library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilised on thesolid surface are so-called ‘bridged’ structures formed by annealing ofpairs of immobilised polynucleotide strands and immobilisedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for nucleic acid sequencing, since hybridisationof a conventional sequencing primer to one of the immobilised strands isnot favoured relative to annealing of this strand to its immobilisedcomplementary strand under standard conditions for hybridisation.

In order to provide more suitable templates for nucleic acid sequencingit is preferred to remove or displace substantially all or at least aportion of one of the immobilised strands in the ‘bridged’ structure inorder to generate a template which is at least partiallysingle-stranded. The portion of the template which is single-strandedwill thus be available for hybridisation to a sequencing primer. Theprocess of removing all or a portion of one immobilised strand in a‘bridged’ double-stranded nucleic acid structure may be referred toherein as ‘linearisation’.

Bridged template structures may be linearised by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease (for example‘USER’, as supplied by NEB, part number M5505S), or by exposure to heator alkali, cleavage of ribonucleotides incorporated into amplificationproducts otherwise comprised of deoxyribonucleotides, photochemicalcleavage or cleavage of a peptide linker.

It will be appreciated that a linearization step may not be essential ifthe solid-phase amplification reaction is performed with only one primercovalently immobilised and the other in free solution.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove or displace the portion(s) of the cleavedstrand(s) that are not attached to the solid support. Suitabledenaturing conditions, for example sodium hydroxide solution, formamidesolution or heat, will be apparent to the skilled reader with referenceto standard molecular biology protocols (Sambrook et al., 2001,Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor Laboratory Press, NY; CurrentProtocols, eds Ausubel et al.). Denaturation results in the productionof a sequencing template which is partially or substantiallysingle-stranded. A sequencing reaction may then be initiated byhybridisation of a sequencing primer to the single-stranded portion ofthe template.

Thus, the invention encompasses methods wherein the nucleic acidsequencing reaction comprises hybridising a sequencing primer to asingle-stranded region of a linearised amplification product,sequentially incorporating one or more nucleotides into a polynucleotidestrand complementary to the region of amplified template strand to besequenced, identifying the base present in one or more of theincorporated nucleotide(s) and thereby determining the sequence of aregion of the template strand.

One preferred sequencing method which can be used in accordance with theinvention relies on the use of modified nucleotides having removable 3′blocks, for example as described in WO04018497 and U.S. Pat. No.7,057,026, the contents of which are incorporated herein by reference intheir entirety. Once the modified nucleotide has been incorporated intothe growing polynucleotide chain complementary to the region of thetemplate being sequenced there is no free 3′-OH group available todirect further sequence extension and therefore the polymerase can notadd further nucleotides. Once the nature of the base incorporated intothe growing chain has been determined, the 3′ block may be removed toallow addition of the next successive nucleotide. By ordering theproducts derived using these modified nucleotides it is possible todeduce the DNA sequence of the DNA template. Such reactions can be donein a single experiment if each of the modified nucleotides has adifferent label attached thereto, known to correspond to the particularbase, to facilitate discrimination between the bases added during eachincorporation step. Alternatively, a separate reaction may be carriedout containing each of the modified nucleotides separately.

The modified nucleotides may comprise a label to facilitate theirdetection. In a particular embodiment, this is a fluorescent label. Eachnucleotide type may comprise a different fluorescent label, for exampleas described in U.S. Provisional Application No. 60/801,270 (WO07135368)(Novel dyes and the use of their labelled conjugates). The detectablelabel need not, however, be a fluorescent label. Any label can be usedwhich allows the detection of an incorporated nucleotide.

One method for detecting fluorescently labelled nucleotides comprisesusing laser light of a wavelength specific for the labelled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means. Suitable instrumentation for recordingimages of clustered arrays is described in U.S. Provisional ApplicationNo. 60/788,248 (WO07123744) (Systems and devices for sequence bysynthesis analysis).

The invention is not intended to be limited to use of the sequencingmethod outlined above, as essentially any sequencing methodology whichrelies on successive incorporation of nucleotides into a polynucleotidechain can be used. Suitable alternative techniques include, for example,Pyrosequencing™, FISSEQ (fluorescent in situ sequencing), MPSS andsequencing by ligation-based methods, for example as described is U.S.Pat. No. 6,306,597.

Sequencing data obtained from each array, whether on a sample pooledafter the amplification reaction such that the treated and untreatedportions are sequenced on the same array, or on separate arrays for eachportion, will reveal which cytosine bases have been converted to uracilbases, and therefore which bases in the sample contained a methylatedcytosine that is resistant to conversion. Analysis of the sequence readsacross the whole nucleic acid sample will, therefore, provide a pictureof the global methylation status of essentially every cytosine base inthe sample.

Kits

The invention also relates to kits for use in methylation analysis usingthe method of the first aspect of the invention.

Preferred embodiments of the kit comprise at least a supply of auniversal methylated mismatch adaptor as defined herein, plus a supplyof at least one amplification primer which is capable of annealing tothe adaptor and priming synthesis of an extension product, whichextension product would include any target sequence ligated to theadaptor when the adaptor is in use.

The particular features of the “mismatch” adaptors for inclusion in thekit are as described elsewhere herein in relation to other aspects ofthe invention, including structures of the forked adaptors. Thestructure and properties of appropriate amplification primers are wellknown to those skilled in the art. Suitable primers of appropriatenucleotide sequence for use with the adaptors included in the kit can bereadily prepared using standard automated nucleic acid synthesisequipment and reagents in routine use in the art. The kit may include asupply of one single type of primer or separate supplies (or even amixture) of two different primers, for example, a pair of PCR primerssuitable for PCR amplification of templates modified with the universaladaptor in solution phase and/or on a suitable solid support.

In one embodiment, the kit may include supplies of differentprimer-pairs for use in solution phase and solid phase PCR. In thiscontext the “different” primer-pairs may be of substantially identicalnucleotide sequence but differ with respect to some other feature ormodification, such as for example surface-capture moieties, etc. Inother embodiments, the kit may include a supply of primers for use in aninitial primer extension reaction and a different primer-pair (or pairs)for solution and/or solid phase PCR amplification.

Adaptors and/or primers may be supplied in the kits ready for use, or asconcentrates requiring dilution before use, or even in a lyophilised ordried form requiring reconstitution prior to use. If required, the kitsmay further include a supply of a suitable diluent for dilution orreconstitution of the primers. Optionally, the kits may further comprisesupplies of reagents, buffers, enzymes, dNTPs, etc. for use in carryingout PCR amplification. Suitable (but non-limiting) examples of suchreagents are as described in the Materials and Methods sections of theaccompanying Examples. Further components which may optionally besupplied in the kit include flow cells for cluster preparation and“universal” sequencing primers suitable for sequencing templatesprepared using the universal adaptors and primers.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

EXAMPLES

In one embodiment of the invention, the genomic DNA is fragmented bylimited digestion with a methylation-insensitive restriction enzymerather than by random hydrodynamic shearing. After size fractionation onan agarose gel, a narrow size window is isolated which constitutes onlya small portion of the genome. By careful size-selection, essentiallythe same genomic subfraction can be isolated from different inputsamples and compared by sequencing. This reduced representation approachhas been used for comparative sequencing (SNP detection; Altshuler etal., Nature 407:513-516 (2000)) and has been proposed as ahigh-throughput method for comparative DNA methylation analysis(Meissner et al., Nucleic Acids Res. 33:5868-5877, 2005). In theexemplary embodiment illustrated in FIG. 10 a, genomic DNA from 4different mouse cell types is digested with the restriction enzyme MspIand size selected to 40-220 bp resulting in a reduced representation ofthe mouse genome that is enriched for CpG dinucleotides and CpG islands.As shown in FIG. 10 b, the size selected fragments are equipped with theaforementioned methylated forked adapters, bisulfite converted andsequenced as described elsewhere in this document. After mapping thebisulfite sequencing reads by aligning them to the mouse referencegenome, methylated cytosines are displayed as bisulfite-resistant Cs.The MspI Reduced Representation Bisulfite Sequencing approach has beenused for comparative methylation profiling of four different mouse celltypes resulting in redundant coverage of almost one million distinct CpGdinucleotides in each cell type with more than 800,000 CpGs covered inall four cell types (FIG. 10 c).

MspI RRBS library construction. Ten μg mouse genomic DNA was digestedwith 100 U of MspI (NEB) in a 500 μl reaction overnight at 37° C.Digested DNA was phenol extracted, ethanol precipitated and sizeselected on a 4% NuSieve 3:1 Agarose gel (Lonza). DNA marker lanes wereexcised from the gel and stained with SYBR Green (Invitrogen). For eachsample, two slices containing DNA fragments of 40-120 bp and 120-220 bp,respectively, were excised from the unstained preparative portion of thegel. DNA was recovered using Easy Clean DNA spin filters (Primm labs,Boston, Mass., USA), phenol extracted and ethanol precipitated. The twosize fractions were kept apart throughout the procedure including thefinal sequencing. Size-selected MspI fragments were filled in and3′-terminal A extended in a 50 μl reaction containing 20 U Klenow exo⁻(NEB), 0.4 mM DATP, 0.04 mM dGTP, and 0.04 mM 5-methyl-dCTP (Roche) in1×NEB buffer 2 (15 min at room temperature followed by 15 min at 37°C.), phenol extracted and ethanol precipitated with 10 μg glycogen(Roche) as a carrier. Ligation to pre-annealed Illumina adapterscontaining 5′-methyl-cytosine instead of cytosine (Illumina) wasperformed using the Illumina DNA preparation kit and protocol. QIAquick(QIAGEN) cleaned-up, adapter-ligated fragments were bisulfite-treatedusing the EpiTect Bisulfite Kit (QIAGEN) with minor modifications: Thebisulfite conversion time was increased to approximately 14 hours byadding 3 cycles (5 min of denaturation at 95° C. followed by 3 hours at60° C.). After bisulfite conversion, the single-strandeduracil-containing DNA was eluted in 20 μl of EB buffer. Analytical (25μl) PCR reactions containing 0.5 μl of bisulfite-treated DNA, 5 pmoleach of genomic PCR primers 1.1 and 2.1 (Illumina) and 2.5 U PfuTurboCxHotstart DNA polymerase (Stratagene) were set up to determine theminimum number of PCR cycles required to recover enough material forsequencing. Preparative scale (8×25 μl) PCR was performed using the samePCR profile: 5 min at 95° C., n×(30 s at 95° C., 20 s at 65° C., 30 s at72° C.) followed by 7 min at 72° C., with n ranging from 18 to 24cycles. QIAquick purified PCR products were subjected to a final sizeselection on a 4% NuSieve 3:1 Agarose gel. SYBR Green-stained gel slicescontaining adapter-ligated fragments of 130-210 bp or 210-310 bp in sizewere excised. RRBS library material was recovered from the gel(QIAquick) and sequenced on an Illumina 1G Genome Analyzer.

The sequences of the relevant adapters are as follows, where everycytosine base contains the 5-methyl group:

(SEQ ID NO. 1) 5′P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG (SEQ ID NO. 2)5′ACACTCTTTCCCTACACGACGCTCTTCCGATCT

The sequences of the two PCR primers are as follows:

(SEQ ID NO. 3)5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO.4) 5′CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

The 3′ terminal residues of these sequences comprise a phosphorothioatelinkage. The oligonucleotides are exonucleases treated with ExonucleaseI and HPLC purified as described below:

Exonuclease I (E. coli) NEB M0293S 20,000 Units/mlExonuclease I storage conditions:100 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 5 mM2-mercaptoethanol, 100 μg/ml BSA and 50% glycerol

1× Exonculease Reaction Buffer 67 mM Glycine-KOH 6.7 mM MgCl₂

10 mM 2-mercaptoethanol

(pH 9.5@25° C.)

Protocol: DNA Primers with a phosphorothioate at the n−1 position (5×85ul of each Primer (approx 25 μM) were aliquoted into Eppendorf tubes. 10μl of 10× Exonuclease I Reaction Buffer and 5 μl of Exonuclease I wasadded to each tube. Each Eppendorf tube was placed in a rack and storedin an oven set at 37° C. for 16 hours. After 16 hr, the tubes wereplaced on a hotblock set at 80° C. for 2 minutes. Then the solutionsfrom the Eppendorfs were passed through P6 Bio Rad columns and spun in acentrifuge at 2000 rpm for 2 minutes. An extra 20 μl of H₂0 was addedand the columns respun. The filtered solutions were placed into aspeedvac and evaporated until each was at 20 μl, and the fractionscombined. The pooled fractions were injected into a reverse phase HPLCsystem, and the main peak was collected. The collected fractions wereevaporated to dryness in a speedvac, 50 μl of water was added and thefraction was subjected again to evaporation to dryness. The resultingpellets were dissolved in 50 μl of water, pooled and the UV measurementtaken to determine the concentration of the oligonucleotide.

The samples were used to isothermally amplify clusters according to themethods below:

Cluster creation was carried out using an Illumina Cluster Station. Toobtain single stranded templates, adapted DNA was first denatured inNaOH (to a final concentration of 0.1N) and subsequently diluted in cold(4° C.) hybridisation buffer (5×SSC+0.05% Tween 20) to workingconcentrations of 2-4 μM, depending on the desired cluster density/tile.120 μl of each sample was primed through each lane of a Solexa flowcell(60 μl/min) mounted on a Solexa Cluster Station, upon which allsubsequent steps are performed. The temperature was ramped to 95° C. for60 s and slowly decreased to 40° C. at a rate of 0.05° C./sec to enableannealing to complementary adapter oligonucleotides immobilised on theflowcell surface (oligo A:5′-PS-TTTTTTTTTT-(diol)3-AATGATACGGCGACCACCGA-3′ (SEQ ID NO. 5); oligoB: 5′-PS-TTTTTTTTTTCAAGCAGAAGACGGCATACGA-3′ (SEQ ID NO. 6)). Hybridisedtemplate strands were extended using Taq polymerase (0.25 U/μl, 200 uMdNTP) in 1× amplification premix (20 mM Tris pH 8.8, 10 mM (NH4)₂SO4, 2mM MgSO₄, 0.1% Triton X-100, 2 M betaine) to generate theirsurface-bound complement. The samples were then denatured usingformamide and washed with wash buffer (0.3×SSC) to remove the initialseeded template. The remaining single stranded copy was the startingpoint for cluster creation. Clusters were amplified under isothermalconditions at 60° C. for 30 cycles using successive rounds ofamplification premix mix (28 μl, 15 μl/min), amplification mix (28 μl at15 μl/min 0.08 U/μl Bst polymerase+200 uM dNTPs in 1× amplificationpremix) and formamide (36 μl at 15 μl/min). Following amplification,clusters were washed with storage buffer (5×SSC). At this stage,clusters were either stored at 4° C. until required for sequencing orimmediately prepared for sequencing.

Linearisation of surface-immobilised complementary oligo-A was achievedby incubation with linearization mix (100 mM sodium periodate, 10 mM3-aminopropan-1-ol, 20 mM Tris pH 8.0, 50% V/V formamide) for 20 minutesat 20° C. followed by a water wash. All exposed 3′-OH termini of DNA,either from the extended template or unextended surface oligonucleotideswere blocked by dideoxy chain termination using a terminal transferase(0.25 U/μl, 2.4 uM ddNTP, 50 mM potassium acetate, 20 mM Tris acetate,10 mM magnesium acetate, 1 mM dithiothreitol pH 7.9, 37° C., 30 minuteincubation). Linearised and blocked clusters were denatured with 0.1NNaOH prior to hybridisation of the sequencing primer (0.5 uM inhybridisation buffer, sequence=5′ ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQID No. 7)). Processed flowcells were transferred to the Illumina GenomeAnalyser for sequencing.

All processes were conducted as described in the Illumina GenomeAnalyser operating manual. The flowcell was mounted to the analyser,primed with sequencing reagents: position #1=incorporation mix (1 uM NTPmix, 0.015 μg/ml SBS polymerase, 50 mM Tris pH 9.0, 50 mM NaCl, 6 mMMgSO4, 1 mM EDTA, 0.05% Tween 20); position #2=spare (MilliQ wateronly); position #3=scan mix (100 mM Tris pH 7.0, 50 mM sodiumacsorbate); position #4=High salt wash (5×SSC, 0.05% Tween 20); position#5=incorporation buffer (50 mM Tris pH 9.0, 50 mM NaCl, 1 mM EDTA, 0.05%Tween 20); position #6=cleavage mix (100 mM TCEP, 100 mM Tris pH 9.0,100 mM NaCl, 50 mM sodium ascorbate, 0.05% Tween 20); position#7=cleavage buffer (100 mM Tris pH 9.0, 100 mM NaCl, 0.05% Tween 20);position #8=spare. Flowcells were sequenced using standard sequencingrecipes for 37-cycle experiments. Data was analysed using the standardanalysis pipeline.

Each cycle of the sequencing recipe is as follows: Sequencing of theclusters from the above illustrative protocol was carried out usingmodified nucleotides prepared as described in International patentapplication WO 2004/018493, and labeled with four spectrally distinctfluorophores, as described in PCT application number PCT/GB2007/001770,published as WO07135368. Sequencing of clusters is described in moredetail in patent WO06064199. The contents of the above-listed documentsare incorporated herein by reference in their entireties.

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) (SBS polymerase) was used for thenucleotide incorporation steps.

Incorporation: Prime with Incorporation buffer, 125 μL/channel; 60μL/minutes, Heat to 60° C.

Treat with Incorporation mix, 75 μL/channel; 60 μL/minutes. Wait for atotal of 15 minutes in addition to pumping fresh Incorporation mix, 25μL/channel; 60 μL/minutes, every 4 minutes.

Cool to 20° C.

Wash with Incorporation buffer, 75 μL/channel; 60 μL/minutes.Wash with 5×SSC/0.05% Tween 20, 75 μL/channel; 60 μL/minutesPrime with imaging buffer, 100 μL/channel; 60 μL/minutesScan in 4 colors at RT.

Cleavage: Prime with Cleavage buffer (0.1M Tris pH 7.4, 0.1M NaCl and0.05% Tween 20), 125 μL/channel; 60 μL/minutes.

Heat to 60° C.

Treat the clusters with Cleavage mix (100 mM TCEP in Cleavage buffer),75 μL/channel; 60 μL/minutes.Wait for a total of 15 minutes in addition to pumping fresh cleavagemix, 25 μL/channel; 60 μL/minutes, every 4 minutes.

Cool to 20° C.

Wash with Enzymology buffer.Wash with 5×SSC/0.05% Tween 20.

Repeat the process of Incorporation and Cleavage for as many cycles asrequired.

Incorporated nucleotides were detected using the Illumina genomeanalyzer, a Total Internal Reflection based fluorescent CCD imagingapparatus described in “Systems and Devices for Sequence by SynthesisAnalysis,” U.S. Ser. No. 60/788,248, filed Mar. 31, 2006 andcorresponding PCT application PCT/US07/07991, published as WO07123744,the contents of which are incorporated herein by reference in theirentirety.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1. A method of analysing methylation status of cytosine bases in anucleic acid, comprising: a. providing a sample of fragmented doublestranded nucleic acid target fragments derived from said nucleic acid;b. ligating universal adaptors to the fragmented double stranded nucleicacid target fragments to produce adaptor-ligated double stranded nucleicacid target fragments comprising identical nucleic acid bases at eachtermini, wherein cytosine bases in said universal adaptors aremethylated and said universal adaptors comprise a region of doublestranded nucleic acids and at least one region of single strandednucleic acids; c. treating the adaptor-ligated double stranded nucleicacid target fragments with a reagent that converts the non-methylatedcytosine bases to uracil to produce a treated sample of adaptor-ligateddouble stranded nucleic acid target fragments; d. sequencing the treatedadaptor-ligated double stranded nucleic acid target fragments; e.analysing the sequences of the treated sample to determine whichcytosine bases were converted to uracil bases, thereby determining themethylation status of the nucleic acid.
 2. The method according to claim1, wherein said reagent comprises bisulfite.
 3. The method according toclaim 1, wherein the method comprises an additional step ofamplification in solution prior to the sequencing step.
 4. The methodaccording to claim 3, wherein said amplification uses two or moreamplification primers, at least one of said amplification primerscomprising a region that extends beyond the 5′ end of the at least oneregion of single stranded nucleic acids of the universal adaptorsequences.
 5. The method according to claim 3, wherein saidamplification uses two or more amplification primers, at least one ofsaid amplification primers comprising a region that hybridises to the atleast one region of single stranded nucleic acids of the universaladaptor.
 6. The method according to claim 5, wherein said amplificationuses two or more amplification primers, at least one of saidamplification primers comprising a region that hybridises to, andextends beyond the 5′ end of the at least one region of single strandednucleic acids of the universal adaptor.
 7. The method according to claim3, wherein the amplification is a polymerase chain reaction andamplification products of the polymerase chain reaction are collected toprovide a 5′ and 3′ modified library of template polynucleotidemolecules comprising known sequences at their 5′ and 3′ ends.
 8. Themethod according to claim 7, wherein the polymerase chain reaction iscarried out using first oligonucleotide primers capable of annealing toadaptor sequences of each of the adaptor-ligated double stranded nucleicacid target fragments and second oligonucleotide primers capable ofannealing to a region of extended strands produced by extension of thefirst oligonucleotide primers.
 9. The method according to claim 8,wherein the first and second oligonucleotide primers have differentnucleotide sequences.
 10. The method according to claim 9, wherein thefirst and second oligonucleotide primers are capable of annealing to oneof the strands in the region of the double stranded nucleic acids in theadaptor sequences of the adaptor-ligated double stranded nucleic acidtarget fragments.
 11. The method according to claim 1, wherein theuniversal adaptors are forked adaptors formed by annealing partiallycomplementary first methylated and second methylated polynucleotidestrands, wherein a sequence of 5 or more consecutive nucleotides at 3′end of the first strand is complementary to a sequence of 5 or moreconsecutive nucleotides at the 5′ end of the second strand, wherein aduplex region of 5 or more consecutive base pairs is formed by annealingthe first and second strands and wherein a sequence of at least 10consecutive nucleotides at the 5′ end of the first strand and a sequenceof at least 10 consecutive nucleotides at the 3′ end of the secondstrand are not complementary such that a mismatched single strandedregion of at least 10 consecutive nucleotides on each strand remainssingle stranded when the duplex region is annealed.
 12. The methodaccording to claim 11, wherein the duplex region formed when the twostrands are annealed is 5 to 20 consecutive base pairs in length. 13.The method according to claim 11, wherein the mismatched single strandedregion comprises 10 to 50 consecutive unpaired nucleotides on eachstrand.
 14. The method according to claim 1, wherein the nucleic acid isgenomic DNA.
 15. The method according to claim 1 wherein the fragmentsare generated by random shearing.
 16. The method according to claim 15wherein the random shearing is hydroshearing or nebulisation.
 17. Themethod according to claim 1 wherein the fragments are generated using anenzymatic treatment.
 18. The method according to claim 17 wherein theenzymatic treatment is a restriction endonuclease that is selective forCG dinucleotides.
 19. The method according to claim 18 wherein theenzyme is MspI.
 20. The method according to claim 1, wherein thesequencing is performed on a solid support.
 21. The method according toclaim 1, wherein the analysis is performed by comparing the sequence ofthe treated sample against a known reference sequence.
 22. The methodaccording to claim 1, wherein the analysis is performed by comparing thesequence of the treated sample against the sequence of an untreatedsample.
 23. The method according to claim 1, wherein the adaptor-ligateddouble stranded nucleic acid target fragments, or amplicons thereof areimmobilised on a solid support and further amplified prior tosequencing.
 24. The method according to claim 23, wherein the solidsupport is a planar array.
 25. The method according to claim 24, whereinthe planar array is a clustered array of amplified single targetmolecules.
 26. The method according to claim 25, wherein the clusteredarray is formed by solid-phase nucleic acid amplification usingimmobilised amplification primers.
 27. The method according to claim 26,wherein the clustered array is formed by isothermal solid-phase nucleicacid amplification using immobilised amplification primers.
 28. Themethod according to claim 22, wherein the treated and untreated portionsare combined and immobilised on the same support.
 29. The methodaccording to claim 1, wherein said sequencing involves cycles ofaddition of nucleotides.
 30. The method according to claim 29, whereinsaid nucleotides are labelled.
 31. The method according to claim 30,wherein nucleotide labels are fluorophores.
 32. A kit for preparing a 5′and 3′ modified library of template polynucleotide molecules comprisingknown sequences at their 5′ and 3′ ends, the kit comprising methylatedadaptor polynucleotides as defined in claim 1 and oligonucleotideprimers capable of annealing to the methylated adaptor polynucleotides.33. The kit according to claim 32, wherein the oligonucleotide primersare capable of annealing to the at least one region of single strandednucleic acids of the universal adaptor polynucleotides.
 34. The kitaccording to claim 33, wherein the methylated adaptor polynucleotidesare forked adaptors formed by annealing partially complementary firstmethylated and second methylated polynucleotide strands, wherein asequence of 5 or more consecutive nucleotides at 3′ end of the firststrand is complementary to a sequence of 5 or more consecutivenucleotides at the 5′ end of the second strand, wherein a duplex regionof 5 or more consecutive base pairs is formed by annealing the first andsecond strands and wherein a sequence of at least consecutivenucleotides at the 5′ end of the first strand and a sequence of at least10 consecutive nucleotides at the 3′ end of the second strand are notcomplementary such that a mismatched single stranded region of at least10 consecutive nucleotides on each strand remains single stranded whenthe duplex region is annealed.